CN103146835B - Based on method and the test kit of the ELISA test strip food source pathogenic bacterium of NASBA - Google Patents
Based on method and the test kit of the ELISA test strip food source pathogenic bacterium of NASBA Download PDFInfo
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Abstract
The invention discloses method and the test kit of a kind of ELISA test strip based on NASBA food source pathogenic bacterium, belong to technical field of biological.The method comprises the steps: (1) extracts the RNA of food source pathogenic bacterium; (2) design of amplification primers; (3) NASBA reaction is carried out; (4) capturing probe is designed; (5) Nano-Au probe is prepared; (6) Radioactive colloidal gold nucleic acid test strip is prepared; (7) detection of sample.Does the test kit using the method to detect Listeria monocytogenes comprise primer (as SEQ? ID? shown in NO.1 and 2), enzyme, damping fluid, RNaseH, RNA enzyme inhibitors, dNTPs, NTPs, probe is (as SEQ? ID? shown in NO.4,5 and 6) and Radioactive colloidal gold nucleic acid reagent bar etc.NASBA is combined with ELISA test strip by present method, and can realize qualitative or detection by quantitative, cost is low, and detection speed is fast, and specificity is good, highly sensitive, use safety.
Description
Technical field
The invention belongs to technical field of biological, relate to method and the test kit of a kind of ELISA test strip based on NASBA food source pathogenic bacterium.
Background technology
China is the world's strongest nation of food consumption and production, and food safety is great livelihood issues, is the topic that the country and people pays close attention to.But the food safety accident of China frequently occurs in recent years, food-safety problem becomes the nightmare that compatriots can't get rid of already in the heart.According to investigations, the food safety of people's uneasiness is allowed to be the major cause causing common people's Happiness Index lower.Food origin disease is the greatest problem of food safety, brings serious threat to consumer health and safety.Food source pathogenic bacterium are the major virulent factor causing food origin disease.In October, 2011, the miss board Boiled dumplings with "three fresh delicacies" of Shanghai Carrefour hypermarket is detected containing streptococcus aureus, and this bacterium causes pneumonia possibly.In April, 2012, Guangdong has two batches of infant formula powders to be detected containing Enterobacter sakazakii, and this germ can cause serious neonatal meningitis, enterocolitis and septicemia, and mortality ratio is up to more than 50%.In November, 2012, State General Administration for Quality Supervision has organized quality product MOE content courses, and Fujian Province turns round more than 100 times that shrimp taste bar coliform measured value is standard value.Food source pathogenic bacterium are difficult to be monitored timely and effectively always, not only constitute a serious threat to food sanitation and people ' s health, also cause very large impact to foodstuffs industry and national economy.Therefore, perfect, prevention mankind food origin disease the generation of method to foodstuff security system developing a kind of quick, sensitive, special, safe detection food source pathogenic bacterium is significant.
The detection method of traditional food source pathogenic bacterium mainly contains culture identification method, electron microscope observation method, immunological detection method etc.The operation of culture identification method is loaded down with trivial details, takes longer, and additive method has that sensitivity is low, not high, the consuming time length of specificity, need the shortcomings such as expensive instrument.Traditional method limits the rapid detection ability to food source pathogenic bacterium to a great extent, and the molecular biology new technology of development in recent years just can realize high-throughput, detect fast and accurately, wherein nucleic acid amplification technologies makes the sensitivity of Molecular Detection and accuracy greatly improve.In order to the demand making amplification technique more adapt to modern molecular diagnosis, much research all concentrates on the exploitation of new detection technique.ELISA test strip technology is undoubtedly for Molecular Detection provides approach easily.In the recent period, develop rapidly based on the nucleic acid test strip combined with nucleic acid amplification technologies, it is compared with immunity test strip, has that cost is low, applied range, the feature that highly sensitive, accuracy is good.
Rely on amplification of nucleic acid sequences (Nucleic acid sequence-based amplification, NASBA) be a kind of technology being particularly suitable for cloning RNA, be the sensitive isothermal amplification technology guided the primer with T7 promoter sequence by 1, its a large amount of single stranded RNAs produced are easy in test strip, realize quick, accurate, high-sensitivity detection.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of detection quick, sensitive, accurate, easy and simple to handle be combined with ELISA test strip based on NASBA to eat the method for source pathogenic bacterium.
Another object of the present invention is to provide a kind of test kit using aforesaid method to detect Listeria monocytogenes.
Object of the present invention is achieved through the following technical solutions, and based on the method for the ELISA test strip food source pathogenic bacterium of NASBA, specifically comprises the following steps:
(1) preparation of template: the RNA extracting food source pathogenic bacterium.
(2) design and synthesis two is for the primer of the food source pathogenic bacterium conserved sequence that increases: T7 primer and primer 2, and containing in T7 primer can by the T7 promoter sequence of t7 rna polymerase identification.
(3) NASBA reaction: template step (1) prepared, the primer of step (2) design and synthesis carry out NASBA amplified reaction, obtain single stranded RNA product.
(4) design of probe: according to sequences Design three capturing probes of single stranded RNA product, probe 1 and probe 2 are complementary with the two ends of single stranded RNA product respectively, probe 3 and probe 1 complete complementary; Article three, one end of probe is all modified with functional group.
(5) preparation of Nano-Au probe: the probe 1 of synthesis designed in step (4) is connected with nanometer gold and prepares Nano-Au probe, and obtain the Nano-Au probe for embedding with the resuspended Nano-Au probe of embedding damping fluid.
(6) preparation of Radioactive colloidal gold nucleic acid test strip
Radioactive colloidal gold nucleic acid test strip is made up of base plate, sample panel, gold pad, nitrocellulose filter (NC film) and water sucting plate, sample panel, gold is padded, nitrocellulose filter (NC film) and water sucting plate overlap successively and be fixed on (as shown in Figure 5 structure) on base plate and assemble and obtain Radioactive colloidal gold nucleic acid test strip.
Described gold pad is embedded with Nano-Au probe prepared by step (5); Described nitrocellulose filter has the control line (C line) containing Streptavidin and probe 3 and the detection line (T line) containing Streptavidin and probe 2, detection line is near gold pad, and control line is near water sucting plate.
(7) detect: by the RNA product obtained by step (3), (methane amide can destroy the secondary structure of RNA with containing methane amide, thus improve its hybridization efficiency with probe) SSC damping fluid (sodium citrate buffer solution) mix, mixed solution is added drop-wise in the sample panel of Radioactive colloidal gold nucleic acid test strip, drip SSC damping fluid again, read result, detection line and control line all become redness and show have target sequence to exist, namely there are food source to be measured pathogenic bacterium, only have control line to become redness and show there is no food source to be measured pathogenic bacterium.
Food pathogenic described in step (1) comprises Listeria monocytogenes (Listeria monocytogenes), Salmonellas (Salmonella enterica), Escherichia coli O 157: H7(Escherichia coli O157:H7), Shigellae (Shigella.Spp), streptococcus aureus (Staphylococcus aureus), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus) and bacillus cereus (Yersinia enterocolitica) etc.
Food source pathogenic bacterium conserved sequence described in step (2) is preferably variable region sequences or the virulence gene fragment of food source pathogenic bacterium 16S rRNA gene, the invasin gene A(invA as Listeria monocytogenes 16S ribosomal RNA gene (16SrRNA gene), Salmonellas) etc.
T7 promoter sequence described in step (2) is TAATACGACTCACTATAGGGAGA.
The system of the NASBA reaction described in step (3) comprises: template ribonucleic acid, AMV reversed transcriptive enzyme, AMV reverse transcriptase buffer, t7 rna polymerase, t7 rna polymerase damping fluid, RNaseH, RNA enzyme inhibitors, T7 primer, primer 2, dNTPs, NTPs, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) (DMSO) and DEPC(diethylpyrocarbonate) process water; The final concentration of described T7 primer is 300 ~ 500nM; The final concentration of described primer 2 is 300 ~ 500nM.
The condition optimization of the NASBA reaction described in step (3) is the composed as follows of every 25 μ L reaction systems: template ribonucleic acid 1ng/ μ L, T7 primer 400nM, primer 2 400nM, AMV reversed transcriptive enzyme 8U, AMV reverse transcriptase buffer, t7 rna polymerase 36U, RNA enzyme inhibitors 10U, RNaseH5U, the concentration that NTPs2mM(often plants ribonucleotide triphosphate is 2mM), the concentration that dNTPs1mM(often plants deoxyribonucleoside triphosphate is 1mM), the volume percent of DMSO is 10%, BSA final concentration is 0.1 μ g/ μ L, amplification procedure is preferably: preparation mixed liquor A in 65 DEG C of heating 5 minutes, then in 41 DEG C of cooling 5min, adds enzyme mixation, hatches 90min in 41 DEG C, 4 DEG C of termination reactions, described mixed liquor A comprises: template ribonucleic acid, T7 primer, primer 2, dNTPs, NTPs, 5 × t7 rna polymerase damping fluid, 5 × AMV reverse transcriptase buffer, DMSO and DEPC process water, described enzyme mixation comprises: 5 × t7 rna polymerase damping fluid, 5 × AMV reverse transcriptase buffer, AMV reversed transcriptive enzyme, t7 rna polymerase, RNaseH, RNA enzyme inhibitors, BSA and DEPC process water.
The length of the single stranded RNA product described in step (3) is preferably 120 ~ 270nt.
The length of the probe 1 described in step (4) and probe 2 is preferably 20 ~ 30nt.
The functional group of the probe 1 described in step (4) is preferably sulfydryl, and the functional group of probe 2 and probe 3 is preferably vitamin H.
The particle diameter of the nanometer gold described in step (5) is preferably 13nm.
Embedding damping fluid described in step (5) is: Na
3pO
420mM, BSA(bovine serum albumin) mass percent 5%, Tween X-100 volume percent 0.25%, sucrose mass percent 8%.
The material of the base plate described in step (6) is preferably PVC plastic (igelite).
The material of the sample panel described in step (6) is preferably glass fibre, and its treatment process is: infiltrate with sample panel process damping fluid, be placed in moisture eliminator room temperature preservation; Described sample panel process damping fluid is: pH8.0, the Triton X-100 of volume percent 0.25%, 0.05M Tris-HCl, 0.15M NaCl.
The material of the gold pad described in step (6) is preferably glass fibre, its treatment process for: with described in 50 μ L steps (4) for the Nano-Au probe spray that embeds thereon, dry under room temperature, 4 DEG C of preservations in moisture eliminator.
The treatment process of the nitrocellulose filter described in step (6) is preferably: the position with spray film instrument, 6 μ L solution of streptavidin and probe 2 mixed solution being sprayed onto detection line (T line), 6 μ L solution of streptavidin and probe 3 mixed solution are sprayed onto the position of control line (C line), dry 1h under being placed in room temperature, and in 4 DEG C of kept dry; Described solution of streptavidin concentration is 1.67mg/mL, and described probe 2 and the concentration of probe 3 are 1mM; Described detection live width 2mm pads with gold 6mm of being separated by, and described control live width 2mm pads with gold 12mm of being separated by.
The material of the water sucting plate described in step (6) is preferably water-absorption fiber.
Assembling described in step (6) is preferably: base plate is at orlop, NC film is pasted onto the middle part on base plate, gold pad is positioned at the side on the top of NC film and overlapping 2mm with it, sample panel is positioned at the top overlapping 2mm with it of gold pad, the top that water sucting plate is positioned at NC film is relatively padded with golden and the opposite side of sample panel 2mm overlapping with NC film, is finally cut into the wide bar of 4mm with slitting shear machine.
The process of the detection of the sample described in step (7) is preferably: the solution that 4 × SSC damping fluid that the RNA product obtained 25 μ L by step (3) and 125 μ L contain volumetric concentration 6% methane amide forms is added drop-wise in the sample panel of Radioactive colloidal gold nucleic acid test strip, after 10min, drip 50 μ L4 × SSC damping fluids again, within 15min, read result.
When described food source pathogenic bacterium are Listeria monocytogenes, its conserved sequence is 16S rRNA gene;
The primer of amplification conserved sequence is:
T7 primer: 5 '-AATTCTAATACGACTCACTATAGGGAGACATCTGTAAGCGATAGCC-3 ';
Primer 2: 5 '-AGCTTGCTCTTCCAA-3 ';
The sequence of single stranded RNA product is: 5 '-
cAUCUGUAAGCGAUAGCCGAAAcCAUCUUUCAAAAGCGUGGCAUGCGCCACACUUUAUCAUUCGGUAUUAGCUCCGGU UUCCCGGAGUUAUCCCCAACUUACAGGCAGGUUGCCCACGUGUUACUCACCCGUCC GC
cACUAACUUUGGAAGAGCAAGCu-3 '; Single underscore part is that what double underline represented is the region that probe 2 combines with probe 1 complementary pairing; Probe 3 and probe 1 complete complementary;
Probe 1 is: 5 '-SH-GCTTGCTCTTCCAAAGTTAGTG-3 ' (SH represents sulfydryl);
Probe 2 is: 5 '-TTTCGGCTATCGCTTACAGATG-Bio-3 ' (Bio represents vitamin H);
Probe 3 is: 5 '-Bio-CACTAACTTTGGAAGAGCAAGC-3 ' (Bio represents vitamin H).
Use aforesaid method to detect the test kit of Listeria monocytogenes, comprise A, B two points of test kits;
A divides test kit to comprise primer, enzyme, enzyme buffer liquid, RNaseH, RNA enzyme inhibitors, bovine serum albumin (BSA), dNTPs, NTPs, DMSO, DEPC process water, wherein:
Described primer comprises T7 primer and primer 2:
T7 primer: 5 '-AATTCTAATACGACTCACTATAGGGAGACATCTGTAAGCGATAGCC-3 ';
Primer 2: 5 '-AGCTTGCTCTTCCAA-3 ';
Described enzyme comprises AMV reversed transcriptive enzyme and t7 rna polymerase;
Described enzyme buffer liquid comprises: AMV reverse transcriptase buffer, T7RNA enzyme buffer liquid;
B divides test kit to comprise probe, damping fluid, Radioactive colloidal gold nucleic acid reagent bar;
Described probe comprises probe 1,2 and 3:
Probe 1 is: 5 '-SH-GCTTGCTCTTCCAAAGTTAGTG-3 ' (SH represents sulfydryl);
Probe 2 is: 5 '-TTTCGGCTATCGCTTACAGATG-Bio-3 ' (Bio represents vitamin H);
Probe 3 is: 5 '-Bio-CACTAACTTTGGAAGAGCAAGC-3 ' (Bio represents vitamin H);
Described damping fluid comprises 4 × SSC damping fluid, 4 × SSC damping fluid containing volumetric concentration 6% methane amide.
Described Radioactive colloidal gold nucleic acid reagent bar comprises and is attached to base plate (material is PVC plastic) upper and the sample panel overlapped successively (material is glass fibre), gold pad (material is glass fibre), nitrocellulose filter and water sucting plate (material is water-absorption fiber); Gold pad is embedded with the Nano-Au probe of linking probe 1; Nitrocellulose filter has the control line (C line) containing Streptavidin and probe 3 and the detection line (T line) containing Streptavidin and probe 2, detection line is near gold pad, and control line is near water sucting plate.
Ultimate principle of the present invention (as shown in Figure 1): when detection system exists goal gene fragment, T7 primer and its combination are annealed, and also there is AMV reversed transcriptive enzyme, RNase H and t7 rna polymerase in system simultaneously.Under the effect of AMV reversed transcriptive enzyme, form DNA-RNA heterozygote, the RNaseH then in system is by the RNA chain degrade specifically in heterozygote.Primer 2 is single stranded DNA combination annealing therewith subsequently, because AMV reversed transcriptive enzyme has the DNA polymerase activity of DNA dependence, therefore under the effect of AMV reversed transcriptive enzyme, catalyzes and synthesizes double-stranded DNA.5 ' the end band because of T7 primer has can by the promoter sequence of t7 rna polymerase identification, so this double-stranded DNA transcribes synthesize antisense rna with regard to the catalytic substrate that can be used as t7 rna polymerase.Sense-rna can be combined with primer 2 and anneal, under the effect of AMV reversed transcriptive enzyme, catalyze and synthesize RNA-DNA heterozygote, then RNase H degrade specifically RNA chain, the single stranded DNA formed is combined with T7 primer anneals, under the effect of AMV reversed transcriptive enzyme, synthesize the double-stranded DNA with t7 rna polymerase recognition site, transcribe synthesize antisense rna again.Sense-rna recirculation carries out the transcription amplification of DNA synthesis, RNA degraded, t7 rna polymerase, and RNA is constantly increased.When the RNA product of amplification gained is added drop-wise in sample panel, by chromatography effect, the Nano-Au probe that first and golden pad place has embedded is hybridized, when its arrival has been embedded with the detection line of the probe 2 of RNA product complementation (T line) place, RNA, Nano-Au probe and probe 2 is formed " sandwich structure ", will form a red line, excessive Nano-Au probe continues chromatography, arrives the line forming Article 2 redness when being embedded with control line (C line) place of probe 3.
And during not containing goal gene fragment, detection line (T line) place is not because forming the hybrid product of " sandwich " structure, and do not have red line to occur, only have excessive Nano-Au probe to continue chromatography, arrive and when being embedded with control line (C line) place of probe 3, to form a red line.
If detection line (T line) and control line (C line) place all redfree line occur, then show that test strip lost efficacy, detect unsuccessfully, sample needs again to detect.
The present invention has following advantage and effect relative to prior art:
(1) relying on amplification of nucleic acid sequences (NASBA) can by template ribonucleic acid amplification 10 in 2h
9doubly, higher than Standard PCR method 1000 times.NASBA technology due on its primer with T7 promoter sequence, and external double-stranded DNA is without T7 promoter sequence, can not be amplified, and therefore under the condition having DNA pollution, NASBA technology has higher specificity and sensitivity equally.Transcriptive process,reversed is directly merged in amplified reaction by NASBA, shortens the reaction times.
(2) be combined with ELISA test strip by NASBA, can realize qualitative or detection by quantitative, result is stablized.
(3) detection speed is fast, and specificity is good, highly sensitive.
(4) equipment is simple, and cost is low, without the need to the instrument of costliness.
(5) probe design is simple, and operation steps is brief, is easy to promote.
(6) the present invention does not have the use of the bromination objectionable impurities such as ingot, isotropic substance, does not have potential safety hazard, use safety.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the ELISA test strip food-borne pathogens based on NASBA.
Fig. 2 is the agarose gel electrophoresis figure of Listeria monocytogenes total serum IgE.
Fig. 3 is the agarose gel electrophoresis figure of NASBA reaction product.
Fig. 4 is the abosrption spectrogram of 13nm Radioactive colloidal gold, has maximum absorption band at 520mm place.
Fig. 5 is packaging assembly and each several part embedding situation map of test strip.
Fig. 6 is the ELISA test strip Listeria monocytogenes result figure based on NASBA, negative findings (No. 1 test strip) and positive findings (No. 2 test strip).
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Method of the present invention is applied to the detection of Listeria monocytogenes.Be illustrated in figure 1 the schematic diagram of the ELISA test strip food-borne pathogens based on NASBA.In embodiment, agents useful for same is all purchased from precious biotechnology (Dalian) company limited and Sangon Biotech (Shanghai) Co., Ltd., and test strip material and equipment are all purchased from Shanghai Jinbiao Bio-Tech Co., Ltd..
Embodiment 1
(1) extraction of Listeria monocytogenes (bacterial strain CMCC54007 buys in Guangzhou institute of microbiology) total serum IgE:
1. liquid nitrogen grinding, gets about 5mL bacterium liquid in mortar, adds a small amount of liquid nitrogen, grind rapidly, constantly add liquid nitrogen in process of lapping, until grinding fully.Proceeded in 1.5mLEP pipe by cell lysate after grinding, then in mill, add 0.8mL Trizol, the remaining lysate on piping and druming mortar, pours in EP pipe.
2. build lid, put upside down mixing 10 times, room temperature leaves standstill 5 minutes.
3. in EP pipe, add 0.2mL chloroform, build lid, firmly rocked for 15 seconds, liquid is fully mixed, room temperature left standstill after 5 minutes, and 12000 leave the heart 15 minutes.
4. supernatant is transferred in new 1.5mL EP pipe (being approximately 0.5mL), adds 0.5mL Virahol, firmly rocked for 15 seconds, liquid is fully mixed, to be positioned in-20 DEG C of refrigerators 1 hour.
5. 12000 leave the heart 10 minutes, careful supernatant discarded, add the DEPC solution of 75% alcohol of 1 milliliter of precooling, concussion washing.
6. 7500 leave the heart 5 minutes, careful supernatant discarded, uncap and dry up precipitation in 30 minutes in super clean bench, now RNA precipitation becomes transparent.
7. add in EP pipe 20 μ L DEPC solubilize precipitation, as resolution of precipitate difficulty, can in 55 ~ 60 DEG C of water-baths maximum 10 minutes hydrotropies.RNA after extraction can be positioned in-80 DEG C of refrigerators and preserve or test immediately.
Extract result as shown in Figure 2, M is DL2000, and 1 is Listeria monocytogenes total serum IgE, comprises the rRNA that 3 kinds of settling ratios are different in the ribosomal subunit of bacterium, is 16S rRNA, 5S rRNA and 23S rRNA respectively.The abundance of all the other RNA is low, in agarose electrophoresis, do not see band.
(2) primer and probe design: the 16S rRNA gene variable region sequences Design according to Listeria monocytogenes can amplify the RNA fragment of 159nt (as shown in Figure 3, M is DL2000, and 1,2 are respectively the amplified band under 1ng/ μ L, 0.1ng/ μ L template concentrations) primer (T7 primer and primer 2).Probe 1 and probe 2 are complementary with the two ends of RNA product respectively, probe 3 and probe 1 complete complementary.Used primer and probe as shown in table 1:
Table 1 list of primers
(3) NASBA reaction:
1. in PCR pipe, prepare following mixed solution: template ribonucleic acid 1 μ L, T7 primer and each 1 μ L, the dNTPs(10mM of primer 2 (10 μMs)) 2.5 μ L, NTPs(10mM) 5 μ L, 5 × t7 rna polymerase damping fluid 2 μ L, 5 × AMV reverse transcriptase buffer 2 μ L, DMSO2.5 μ L, DEPC process water 3 μ L, cumulative volume 20 μ L;
2. 65 DEG C hatch 5 minutes after, 41 DEG C cooling 5min;
3. in above-mentioned PCR pipe, following enzyme mixation (5 μ L) is added rapidly: 5 × t7 rna polymerase damping fluid 0.5 μ L, 5 × AMV reverse transcriptase buffer 0.5 μ L, AMV reversed transcriptive enzyme (10U/ μ L) 0.8 μ L, t7 rna polymerase (20U/ μ L) 1.8 μ L, RNaseH(5U/ μ L) 0.1 μ L, RNA enzyme inhibitors (40U/ μ L) 0.25 μ L, BSA(10 μ g/ μ L) 0.25 μ L, DEPC process water 0.8 μ L;
4. hatch 90 minutes for 41 DEG C, 4 DEG C of termination reactions, the product obtained can be directly used in ELISA test strip.
(4) preparation of nanometer gold
Adopt citrate reduction legal system for nanometer gold, by the HAuCl of 100mL1mM
4ebuillition of heated, the sodium citrate solution of 10mL38.8mM is added rapidly under rapid stirring, in 2min, solution colour is by the redness of golden yellow → grey → burgundy → bright, continue to stir 20min, be cooled to room temperature and obtain nano Au colloid liquid solution, record its absorption peak at about 520nm place (shown in Fig. 4) with ultra-violet absorption spectrum instrument, namely obtain the nano Au colloid liquid solution of 13nm.
(5) connection (TCEP method marking nano Au probe) of probe 1 and nanometer gold
5 ' the terminal modified probe 1 having sulfydryl of 20 μ L200 μMs is added in PCR pipe, then in PCR pipe, add acetate buffer solution (pH5.2) and the 1 μ L10 μM TCEP(Trichloroethyl Phosphate of 0.66 μ L500 μM) with activated thiol groups, room temperature, lucifuge hatch 1 hour; Get clean centrifuge tube, add the nano Au colloid liquid solution of 1mL10nM, then under constantly shaking, add probe 1 solution of TCEP process, lid upper tube cap, room temperature lucifuge is placed at least 16 hours, with vibrator 600rpm accelerated reaction; In pipe, dropwise add the Tris acetate buffer solution (pH8.2) of 20 μ L500mM under slow concussion, the final concentration of Tris acetate buffer solution is 5mM; In pipe, dropwise add 200 μ L1M NaCl again, lucifuge hatches one day; 12000rpm, 4 DEG C centrifugal 30 minutes, and take out centrifuge tube, nanoparticle is deposited at the bottom of pipe, sops up supernatant gently, embeds damping fluid (20mM Na with 1mL
3pO
4, mass percent 5%BSA, volume percent 0.25%Tween X-100, mass percent 8%sucrose) and resuspended, for the embedding at test strip gold pad place.
(6) assembling of Radioactive colloidal gold nucleic acid test strip and preparation
1. sample panel: infiltrate with sample panel process damping fluid (TritonX-100 of pH8.0, volume percent 0.25%, 0.05M Tris-HCl, 0.15M NaCl), be placed in moisture eliminator room temperature preservation;
2. gold pad: the nanometer gold-probe 1 solution spray marked by 60 μ L is thereon, dry under room temperature, 4 DEG C of preservations in moisture eliminator;
3. NC film: with spray film instrument by the Streptavidin of 6 μ L1.67mg/mL and the terminal modified position having the mixed solution of the probe 2 of vitamin H to be sprayed onto detection line (T line) of 1mM3 ', by the solution of streptavidin of 6 μ L1.67mg/mL and the terminal modified position having the mixed solution of the probe 3 of vitamin H to be sprayed onto control line (C line) of 1mM5 ', C line and T line place scribe widths are 2mm, article two, line is separated by 4mm, dry 1h under the NC film of embedded probe is placed in room temperature, and in 4 DEG C of kept dry;
4. assemble: assemble by structure shown in Fig. 5, base plate is at orlop, NC film is pasted onto the middle part on base plate, the side that gold pad is positioned at the top of NC film is also overlapping with it, the top that sample panel is positioned at gold pad is overlapping with it, and the top that water sucting plate is positioned at nitrocellulose filter is overlapping with nitrocellulose filter relative to the opposite side of gold pad and sample panel; Overlapping 2mm between every part, is finally cut into the bar that 4mm is wide.
(7) preparation of samples and detection
The solution that the RNA product obtained 25 μ L by step (3) and 125 μ L4 × SSC damping fluids (containing volumetric concentration 6% methane amide) form is added drop-wise in the sample panel of Radioactive colloidal gold nucleic acid test strip, after 10min, drip 50 μ L4 × SSC damping fluids again, within 15min, read result.Detected result is as shown in Figure 6: (Listeria monocytogenes is negative for No. 1 test strip, blank) only form red line at C line place, No. 2 test strip (Listeria monocytogenes is positive) all form red line at C line and T line place, show that this test strip can correctly detect whether containing goal gene fragment in sample, and then determine detected food pathogenic.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1., based on a method for the ELISA test strip food source pathogenic bacterium of NASBA, it is characterized in that comprising the following steps:
(1) preparation of template: the RNA extracting food source pathogenic bacterium;
(2) design and synthesis two is for the primer of the food source pathogenic bacterium conserved sequence that increases: T7 primer and primer 2, and containing in T7 primer can by the T7 promoter sequence of t7 rna polymerase identification;
(3) NASBA reaction: template step (1) prepared, the primer of step (2) design and synthesis carry out NASBA amplified reaction, obtain single stranded RNA product;
(4) design of probe: according to sequences Design three capturing probes of single stranded RNA product, probe 1 and probe 2 are complementary with the two ends of single stranded RNA product respectively, probe 3 and probe 1 complete complementary; Article three, one end of probe is all modified with functional group;
(5) preparation of Nano-Au probe: the probe 1 of synthesis designed in step (4) is connected with nanometer gold and prepares Nano-Au probe, and obtain the Nano-Au probe for embedding with the resuspended Nano-Au probe of embedding damping fluid;
(6) preparation of Radioactive colloidal gold nucleic acid test strip
Radioactive colloidal gold nucleic acid test strip is made up of base plate, sample panel, gold pad, nitrocellulose filter and water sucting plate, sample panel, gold pad, nitrocellulose filter and water sucting plate is overlapped successively to be fixed on assembling on base plate and to obtain Radioactive colloidal gold nucleic acid test strip;
Described gold pad is embedded with Nano-Au probe prepared by step (5); Described nitrocellulose filter there are the control line containing Streptavidin and probe 3 and the detection line containing Streptavidin and probe 2;
(7) detect: the RNA product obtained by step (3) is mixed with the sodium citrate buffer solution containing methane amide, mixed solution is added drop-wise in the sample panel of Radioactive colloidal gold nucleic acid test strip, drip sodium citrate buffer solution again, read result, detection line and control line all become redness and show have target sequence to exist, namely there are food source to be measured pathogenic bacterium, only have control line to become redness and show there is no food source to be measured pathogenic bacterium;
Food source pathogenic bacterium described in step (1) are Listeria monocytogenes;
T7 promoter sequence described in step (2) is TAATACGACTCACTATAGGGAGA;
Food source pathogenic bacterium conserved sequence described in step (2) is the variable region sequences of the 16S rRNA gene of Listeria monocytogenes;
The primer of amplification conserved sequence is:
T7 primer: 5 '-AATTCTAATACGACTCACTATAGGGAGACATCTGTAAGCGATAGCC-3 ';
Primer 2: 5 '-AGCTTGCTCTTCCAA-3 ';
Probe 1 is: 5 '-sulfydryl-GCTTGCTCTTCCAAAGTTAGTG-3 ';
Probe 2 is: 5 '-TTTCGGCTATCGCTTACAGATG-vitamin H-3 ';
Probe 3 is: 5 '-vitamin H-CACTAACTTTGGAAGAGCAAGC-3 ';
The method of the described food of the ELISA test strip based on NASBA source pathogenic bacterium is used for non-diagnostic object.
2. the method for the food of the ELISA test strip based on NASBA source according to claim 1 pathogenic bacterium, is characterized in that:
The system of the NASBA reaction described in step (3) comprises: template ribonucleic acid, AMV reversed transcriptive enzyme, AMV reverse transcriptase buffer, T7 RNA polymerase, T7 RNA polymerase damping fluid, RNaseH, RNA enzyme inhibitors, T7 primer, primer 2, dNTPs, NTPs, bovine serum albumin, DMSO and DEPC process water; The final concentration of described T7 primer is 300 ~ 500nM; The final concentration of described primer 2 is 300 ~ 500nM.
3. the method for the food of the ELISA test strip based on NASBA source according to claim 1 pathogenic bacterium, is characterized in that:
The condition of the NASBA reaction described in step (3) is the composed as follows of every 25 μ L reaction systems: the volume percent of template ribonucleic acid 1ng/ μ L, T7 primer 400nM, primer 2 400nM, AMV reversed transcriptive enzyme 8U, AMV reverse transcriptase buffer, T7 RNA polymerase 36U, RNA enzyme inhibitors 10U, RNaseH 5U, NTPs 2mM, dNTPs 1mM, DMSO is 10%, bovine serum albumin final concentration is 0.1 μ g/ μ L; Amplification procedure is: preparation mixed liquor A in 65 DEG C of heating 5 minutes, then in 41 DEG C of cooling 5min, adds enzyme mixation, hatches 90min in 41 DEG C, 4 DEG C of termination reactions; Described mixed liquor A comprises: template ribonucleic acid, T7 primer, primer 2, dNTPs, NTPs, 5 × T7 RNA polymerase damping fluid, 5 × AMV reverse transcriptase buffer, DMSO and DEPC process water; Described enzyme mixation comprises: 5 × T7 RNA polymerase damping fluid, 5 × AMV reverse transcriptase buffer, AMV reversed transcriptive enzyme, T7 RNA polymerase, RNaseH, RNA enzyme inhibitors, bovine serum albumin and DEPC process water.
4. the method for the food of the ELISA test strip based on NASBA source according to claim 1 pathogenic bacterium, is characterized in that:
The particle diameter of the nanometer gold described in step (5) is 13nm;
Embedding damping fluid described in step (5) is: Na
3pO
420mM, BSA mass percent 5%, Triton X-100 volume percent 0.25%, sucrose mass percent 8%.
5. the method for the food of the ELISA test strip based on NASBA source according to claim 1 pathogenic bacterium, is characterized in that:
The material of the base plate described in step (6) is igelite;
The material of the sample panel described in step (6) is glass fibre, and its treatment process is: infiltrate with sample panel process damping fluid, be placed in moisture eliminator room temperature preservation; Described sample panel process damping fluid is: pH 8.0, the Triton X-100 of volume percent 0.25%, 0.05M Tris-HCl, 0.15M NaCl;
The material of the gold pad described in step (6) is glass fibre, its treatment process for: with described in 50 μ L steps (4) for the Nano-Au probe spray that embeds thereon, dry under room temperature, 4 DEG C of preservations in moisture eliminator;
The treatment process of the nitrocellulose filter described in step (6) is: the position with spray film instrument, 6 μ L solution of streptavidin and probe 2 mixed solution being sprayed onto detection line, 6 μ L solution of streptavidin and probe 3 mixed solution are sprayed onto the position of control line, dry 1h under being placed in room temperature, and in 4 DEG C of kept dry; Described solution of streptavidin concentration is 1.67mg/mL, and described probe 2 and the concentration of probe 3 are 1mM;
The material of the water sucting plate described in step (6) is water-absorption fiber;
Being assembled into described in step (6): base plate is at orlop, nitrocellulose filter is pasted onto the middle part on base plate, the side that gold pad is positioned at the top of nitrocellulose filter is also overlapping with it, the top that sample panel is positioned at gold pad is overlapping with it, and the top that water sucting plate is positioned at nitrocellulose filter is relatively padded with golden and the opposite side of sample panel overlapping with nitrocellulose filter.
6. the method for the food of the ELISA test strip based on NASBA source according to claim 1 pathogenic bacterium, is characterized in that:
The process of the detection of the sample described in step (7) is: the solution that 4 × sodium citrate buffer solution that the RNA product obtained 25 μ L by step (3) and 125 μ L contain volumetric concentration 6% methane amide forms is added drop-wise in the sample panel of Radioactive colloidal gold nucleic acid test strip, after 10min, drip 50 μ L 4 × sodium citrate buffer solutions again, within 15min, read result.
7. use the method described in any one of claim 1 ~ 4 to detect the test kit of Listeria monocytogenes, it is characterized in that: described test kit comprises A, B two points of test kits;
A divides test kit to comprise primer, enzyme, enzyme buffer liquid, RNaseH, RNA enzyme inhibitors, bovine serum albumin, dNTPs, NTPs, DMSO, DEPC process water, wherein:
Described primer comprises T7 primer and primer 2:
T7 primer: 5 '-AATTCTAATACGACTCACTATAGGGAGACATCTGTAAGCGATAGCC-3 ';
Primer 2: 5 '-AGCTTGCTCTTCCAA-3 ';
Described enzyme comprises AMV reversed transcriptive enzyme and T7 RNA polymerase;
Described enzyme buffer liquid comprises: AMV reverse transcriptase buffer, T7 RNA enzyme buffer liquid;
B divides test kit to comprise probe, damping fluid, Radioactive colloidal gold nucleic acid reagent bar;
Described probe comprises probe 1,2 and 3:
Probe 1 is: 5 '-sulfydryl-GCTTGCTCTTCCAAAGTTAGTG-3 ';
Probe 2 is: 5 '-TTTCGGCTATCGCTTACAGATG-vitamin H-3 ';
Probe 3 is: 5 '-vitamin H-CACTAACTTTGGAAGAGCAAGC-3 ';
Described damping fluid comprises 4 × sodium citrate buffer solution, 4 × sodium citrate buffer solution containing volumetric concentration 6% methane amide;
Described Radioactive colloidal gold nucleic acid reagent bar comprise be attached on base plate and the sample panel overlapped successively, gold pad, nitrocellulose filter and water sucting plate; Gold pad is embedded with the Nano-Au probe of linking probe 1; Nitrocellulose filter has the control line containing Streptavidin and probe 3 and the detection line containing Streptavidin and probe 2, detection line is near gold pad, and control line is near water sucting plate.
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| CN104673786A (en) * | 2015-03-09 | 2015-06-03 | 武汉格蓝丽富科技有限公司 | Method for selective amplification of RNA |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101636415A (en) * | 2006-12-22 | 2010-01-27 | 拜奥默里克斯公司 | Be used for enrichment, remove and detect the ways and means of gram positive bacterium |
| CN101918593A (en) * | 2007-12-21 | 2010-12-15 | 生物梅里埃股份有限公司 | Detection of methicillin-resistant staphylococcus aureus |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101636415A (en) * | 2006-12-22 | 2010-01-27 | 拜奥默里克斯公司 | Be used for enrichment, remove and detect the ways and means of gram positive bacterium |
| CN101918593A (en) * | 2007-12-21 | 2010-12-15 | 生物梅里埃股份有限公司 | Detection of methicillin-resistant staphylococcus aureus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868223A (en) * | 2017-04-19 | 2017-06-20 | 广州中医药大学 | A kind of asymmetric PCR amplimer of dengue virus NS5 genetic fragments and its application |
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